Voltage-gated Na channels are responsible for initiation and propagation of the action potential in vertebrate nerve and muscle. Because of its essential physiological role in movement, the Na channel is a prime target of paralytic neurotoxins, which act at five or more distinct neurotoxin receptor sites. The genes encoding the polypeptide scorpion toxins have been cloned and successfully expressed in bacteria to produce large amounts of these toxins. Therefore, these toxins constitute a substantial terrorist threat as peptides. Moreover, bacteria or viruses expressing the potent polypeptide scorpion toxins are themselves terrorist threats because infection of human hosts with these agents would result in paralysis. The central hypothesis of the work proposed here is that toxin antagonists can be produced that will protect broadly and effectively against paralytic peptide neurotoxins. This hypothesis is supported by a proof-of-concept from our current research, in which the first antagonist of scorpion toxin action has been produced. In the research proposed here, we will define the receptor sites and mechanisms of action of the a- and [unreadable]-scorpion toxins on Na channels, and we will develop therapeutic agents to prevent their toxic actions as well as the toxic actions of mechanistically related peptide neurotoxins from other sources. Our Specific Aims are: 1. Molecular mapping of the scorpion toxin receptor sites on Na channels. 2. Molecular mapping of the active sites of a- and [unreadable]-scorpion toxins. 3. Three-dimensional models of the scorpion toxin receptor sites. 4. Development of novel and potent toxin and small peptide antagonists. All of our work in Specific Aims 1 through 3 immediately flow into the design and development of toxin antagonists in Specific Aim 4 and will significantly advance the effort to develop novel therapeutic agents to protect against the threat of paralytic neurotoxins. These studies will provide new insights into the molecular mechanisms of toxin action on Na channels and will lead to development of effective antagonists of toxin action. These advances will be of crucial importance to developing an arsenal of counter-terrorism agents to prevent illness and deaths from potential bioterrorist attacks using these potent paralytic neurotoxins. In addition to these important advances for counter-terrorism, these studies will shed new light on the molecular mechanisms of voltage sensing and activation gating of Na channels, an essential step toward understanding the molecular mechanisms of electrical excitability and potentially a novel approach to development of drugs to treat chronic pain and neurological disease. [unreadable] [unreadable] [unreadable]